Humanised antibodies

ABSTRACT

CDR-grafted antibody heavy and light chains comprise acceptor framework and donor antigen binding regions, the heavy chains comprising donor residues at at least one of positions (6, 23) and/or (24, 48) and/or (49, 71) and/or (73, 75) and/or (76) and/or (78) and (88) and/or (91). The CDR-grafted light chains comprise donor residues at at least one of positions (1) and/or (3) and (46) and/or (47) or at at least one of positions (46, 48, 58) and (71). The CDR-grafted antibodies are preferably humanized antibodies, having non human, e.g. rodent, donor and human acceptor frameworks, and may be used for in vivo therapy and diagnosis. A generally applicable protocol is disclosed for obtaining CDR-grafted antibodies.

Notice: More than one reissue application has been filed for the reissueof U.S. Pat. No. 7,556,771. The reissue applications are applicationNos. 16/378,731 (the present application), and 17/464,970, which is areissue continuation of U.S. Pat. No. 7,556,771. This application is areissue of U.S. Pat. No. 7,566,771, which was filed as application Ser.No. 08/485,686 on Jun. 7, 1995, which is a continuation , of applicationSer. No. 08/303,569, filed on Sep. 7, 1994 and issued as U.S. Pat. No.5,859,205, which is a § 1.62 continuation of U.S. application Ser. No.07/743,329, filed Sep. 17, 1991, now abandoned, which is the U.S.counterpart of PCT/GB90/02017 filed Dec. 21, 1990, originally filed aswhich claims the priority benefit of United Kingdom Application SerialNo. 8928874, filed Dec. 21, 1989.

FIELD OF THE INVENTION

The present invention relates to humanised antibody molecules, toprocesses for their production using recombinant DNA technology, and totheir therapeutic uses.

The term “humanised antibody molecule” is used to describe a moleculehaving an antigen binding site derived from an immunoglobulin from anon-human species, and remaining immunoglobulin-derived parts of themolecule being derived from a human immunoglobulin. The antigen bindingsite typically comprises complementarity determining regions (CDRs)which determine the binding specificity of the antibody molecule andwhich are carried on appropriate framework regions in the variabledomains. There are 3 CDRs (CDR1, CDR2 and CDR3) in each of the heavy andlight chain variable domains.

In the description, reference is made to a number of publications bynumber. The publications are listed in numerical order at the end of thedescription.

BACKGROUND OF THE INVENTION

Natural immunoglobulins have been known for many years, as have thevarious fragments thereof, such as the Fab, (Fab')₂ and Fc fragments,which can be derived by enzymatic cleavage. Natural immunoglobulinscomprise a generally Y-shaped molecule having an antigen-binding sitetowards the end of each upper arm. The remainder of the structure, andparticularly the stem of the Y, mediates the effector functionsassociated with immunoglobulins.

Natural immunoglobulins have been used in assay, diagnosis and, to amore limited extent, therapy. However, such uses, especially in therapy,were hindered until recently by the polyclonal nature of naturalimmunoglobulins. A significant step towards the realisation of thepotential of immunoglobulins as therapeutic agents was the discovery ofprocedures for the production of monoclonal antibodies (MAbs) of definedspecificity (1).

However, most MAbs are produced by hybridomas which are fusions ofrodent spleen cells with rodent myeloma cells. They are thereforeessentially rodent proteins. There are very few reports of theproduction of human MAbs.

Since most available MAbs are of rodent origin, they are naturallyantigenic in humans and thus can give rise to an undesirable immuneresponse termed the HAMA (Human Anti-Mouse Antibody) response.Therefore, the use of rodent MAbs as therapeutic agents in humans isinherently limited by the fact that the human subject will mount animmunological response to the MAb and will either remove it entirely orat least reduce its effectiveness. In practice, MAbs of rodent originmay not be used in patients for more than one or a few treatments as aHAMA response soon develops rendering the MAb ineffective as well asgiving rise to undesirable reactions. For instance, OKT3 a mouse IgG2a/kMAb which recognises an antigen in the T-cell receptor-CD3 complex hasbeen approved for use in many countries throughout the world as animmunosuppressant in the treatment of acute allograft rejection[Chatenoud et al (2) and Jeffers et al (3)]. However, in view of therodent nature of this and other such MAbs, a significant HAMA responsewhich may include a major anti-idiotype component, may build up on use.Clearly, it would be highly desirable to diminish or abolish thisundesirable HAMA response and thus enlarge the areas of use of thesevery useful antibodies.

Proposals have therefore been made to render non-human MAbs lessantigenic in humans. Such techniques can be generically termed“humanisation” techniques. These techniques typically involve the use ofrecombinant DNA technology to manipulate DNA sequences encoding thepolypeptide chains of the antibody molecule.

Early method for humanising MAbs involved production of chimericantibodies in which an antigen binding site comprising the completevariable domains of one antibody is linked to constant domains derivedfrom another antibody. Methods for carrying out such chimerisationprocedures are described in EP0120694 (Celltech Limited), EP0125023(Genentech Inc. and City of Hope), EP-A-0 171496 (Res. Dev. Corp.Japan), EP-A-0 173 494 (Stanford University), and WO 86/01533 (CelltechLimited). This latter Celltech application (WO 86/01533) discloses aprocess for preparing an antibody molecule having the variable domainsfrom a mouse MAb and the constant domains from a human immunoglobulin.Such humanised chimeric antibodies, however, still contain a significantproportion of non-human amino acid sequence, i.e. the complete non-humanvariable domains, and thus may still elicit some HAMA response,particularly if administered over a prolonged period [Begent et al (ref.4)].

In an alternative approach, described in EP-A-0239400 (Winter), thecomplementarity determining regions (CDRs) of a mouse MAb have beengrafted onto the framework regions of the variable domains of a humanimmunoglobulin by site directed mutagenesis using long oligonucleotides.The present invention relates to humanised antibody molecules preparedaccording to this alternative approach, i.e. CDR-grafted humanisedantibody molecules. Such CDR-grafted humanised antibodies are much lesslikely to give rise to a HAMA response than humanised chimericantibodies in view of the much lower proportion of non-human amino acidsequence which they contain.

The earliest work on humanising MAbs by CDR-grafting was carried out onMAbs recognising synthetic antigens, such as the NP or NIP antigens.However, examples in which a mouse MAb recognising lysozyme and a ratMAb recognising an antigen on human T-cells were humanised byCDR-grafting have been described by Verhoeyen et al (5) and Riechmann etal (6) respectively. The preparation of CDR-grafted antibody to theantigen on human T cells is also described in WO 89/07452 (MedicalResearch Council).

In Riechmann et al/Medical Research Council it was found that transferof the CDR regions alone [as defined by Kabat refs. (7) and (8)] was notsufficient to provide satisfactory antigen binding activity in theCDR-grafted product. Riechmann et al found that it was necessary toconvert a serine residue at position 27 of the human sequence to thecorresponding rat phenylalanine residue to obtain a CDR-grafted producthaving improved antigen binding activity. This residue at position 27 ofthe heavy chain is within the structural loop adjacent to CDR1. Afurther construct which additionally contained a human serine to rattyrosine change at position 30 of the heavy chain did not have asignificantly altered binding activity over the humanised antibody withthe serine to phenylalanine change at position 27 alone. These resultsindicate that changes to residues of the human sequence outside the CDRregions, in particular in the structural loop adjacent to CDR1, may benecessary to obtain effective antigen binding activity for CDR-graftedantibodies which recognise more complex antigens. Even so the bindingaffinity of the best CDR-grafted antibodies obtained was stillsignificantly less than the original MAb.

Very recently Queen et al (9) have described the preparation of ahumanised antibody that binds to the interleukin 2 receptor, bycombining the CDRs of a murine MAb (anti-Tac) with human immunoglobulinframework and constant regions. The human framework regions were chosento maximise homology with the anti-Tac MAb sequence. In additioncomputer modelling was used to identify framework amino acid residueswhich were likely to interact with the CDRs of antigen, and mouse aminoacids were used at these positions in the humanised antibody.

In WO 90/07861 Queen et al propose four criteria for designing humanisedimmunoglobulins. The first criterion is to use as the human acceptor theframework from a particular human immunoglobulin that is unusuallyhomologous to the non-human donor immunoglobulin to be humanised, or touse a consensus framework from many human antibodies. The secondcriterion is to use the donor amino acid rather than the acceptor if thehuman acceptor residue is unusual and the donor residue is typical forhuman sequences at a specific residue of the framework. The thirdcriterion is to use the donor framework amino acid residue rather thanthe acceptor at positions immediately adjacent to the CDRs. The fourthcriterion is to use the donor amino acid residue at framework positionsat which the amino acid is predicted to have a side chain atom withinabout 3 Å of the CDRs in a three-dimensional immunoglobulin model and tobe capable of interacting with the antigen or with the CDRs of thehumanised immunoglobulin. It is proposed that criteria two, three orfour may be applied in addition or alternatively to criterion one, andmay be applied singly or in any combination.

WO 90/07861 describes in detail the preparation of a single CDR-graftedhumanised antibody, a humanised antibody having specificity for the p55Tac protein of the IL-2 receptor. The combination of all four criteria,as above, were employed in designing this humanised antibody, thevariable region frameworks of the human antibody Eu (7) being used asacceptor. In the resultant humanised antibody the donor CDRs were asdefined by Kabat et al (7 and 8) and in addition the mouse donorresidues were used in place of the human acceptor residues, at positions27, 30, 48, 66, 67, 89, 91, 94, 103, 104, 105 and 107 in the heavy chainand at positions 48, 60 and 63 in the light chain, of the variableregion frameworks. The humanised anti-Tac antibody obtained is reportedto have an affinity for p55 of 3×10⁹ M⁻¹, about one-third of that of themurine MAb.

We have further investigated the preparation of CDR-grafted humanisedantibody molecules and have identified a hierarchy of positions withinthe framework of the variable regions (i.e. outside both the Kabat CDRsand structural loops of the variable regions) at which the amino acididentities of the residues are important for obtaining CDR-graftedproducts with satisfactory binding affinity. This has enabled us toestablish a protocol for obtaining satisfactory CDR-grafted productswhich may be applied very widely irrespective of the level of homologybetween the donor immunoglobulin and acceptor framework. The set ofresidues which we have identified as being of critical importance doesnot coincide with the residues identified by Queen et al (9).

SUMMARY OF THE INVENTION

Accordingly, in a first aspect the invention provides a CDR-graftedantibody heavy chain having a variable region domain comprising acceptorframework and donor antigen binding regions wherein the frameworkcomprises donor residues at at least one of positions 6, 23, and/or 24,48 and/or 49, 71 and/or 73, 75 and/or 76 and/or 78 and 88 and/or 91.

In preferred embodiments, the heavy chain framework comprises donorresidues at positions 23, 24, 49, 71, 73 and 78 or at positions 23, 24and 49. The residues at positions 71, 73 and 78 of the heavy chainframework are preferably either all acceptor or all donor residues.

In particularly preferred embodiments the heavy chain frameworkadditionally comprises donor residues at one, some or all of positions6, 37, 48 and 94. Also it is particularly preferred that residues atpositions of the heavy chain framework which are commonly conservedacross species, i.e. positions 2, 4, 25, 36, 39, 47, 93, 103, 104, 106and 107, if not conserved between donor and acceptor, additionallycomprise donor residues. Most preferably the heavy chain frameworkadditionally comprises donor residues at positions 2, 4, 6, 25, 36, 37,39, 47, 48, 93, 94, 103, 104, 106 and 107.

In addition the heavy chain framework optionally comprises donorresidues at one, some or all of positions:

1 and 3,

72 and 76,

69 (if 48 is different between donor and acceptor),

38 and 46 (if 48 is the donor residue),

80 and 20 (if 69 is the donor residue),

67,

82 and 18 (if 67 is the donor residue),

91,

88, and

any one or more of 9, 11, 41, 87, 108, 110 and 112.

In the first and other aspects of the present invention reference ismade to CDR-grafted antibody products comprising acceptor framework anddonor antigen binding regions. It will be appreciated that the inventionis widely applicable to the CDR-grafting of antibodies in general. Thus,the donor and acceptor antibodies may be derived from animals of thesame species and even same antibody class or sub-class. More usually,however, the donor and acceptor antibodies are derived from animals ofdifferent species. Typically the donor antibody is a non-human antibody,such as a rodent MAb, and the acceptor antibody is a human antibody.

In the first and other aspects of the present invention, the donorantigen binding region typically comprises at least one CDR from thedonor antibody. Usually the donor antigen binding region comprises atleast two and preferably all three CDRs of each of the heavy chainand/or light chain variable regions. The CDRs may comprise the KabatCDRs, the structural loop CDRs or a composite of the Kabat andstructural loop CDRs and any combination of any of these. Preferably,the antigen binding regions of the CDR-grafted heavy chain variabledomain comprise CDRs corresponding to the Kabat CDRs at CDR2 (residues50-65) and CDR3 (residues 95-100) and a composite of the Kabat andstructural loop CDRs at CDR1 (residues 26-35).

The residue designations given above and elsewhere in the presentapplication are numbered according to the Kabat numbering [refs. (7) and(8)]. Thus the residue designations do not always correspond directlywith the linear numbering of the amino acid residues. The actual linearamino acid sequence may contain fewer or additional amino acids than inthe strict Kabat numbering corresponding to a shortening of, orinsertion into, a structural component, whether framework or CDR, of thebasic variable domain structure. For example, the heavy chain variableregion of the anti-Tac antibody described by Queen et al (9) contains asingle amino acid insert (residue 52a) after residue 52 of CDR2 and athree amino acid insert (residues 82a, 82b and 82c) after frameworkresidue 82, in the Kabat numbering. The correct Kabat numbering ofresidues may be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The invention also provides in a second aspect a CDR-grafted antibodylight chain having a variable region domain comprising acceptorframework and donor antigen binding regions wherein the frameworkcomprises donor residues at at least one of positions 1 and/or 3 and 46and/or 47. Preferably the CDR grafted light chain of the second aspectcomprises donor residues at positions 46 and/or 47.

The invention also provides in a third aspect a CDR-grafted antibodylight chain having a variable region domain comprising acceptorframework and donor antigen binding regions wherein the frameworkcomprises donor residues at at least one of positions 46, 48, 58 and 71.

In a preferred embodiment of the third aspect, the framework comprisesdonor residues at all of positions 46, 48, 58 and 71.

In particularly preferred embodiments of the second and third aspects,the framework additionally comprises donor residues at positions 36, 44,47, 85 and 87. Similarly positions of the light chain framework whichare commonly conserved across species, i.e. positions 2, 4, 6, 35, 49,62, 64-69, 98, 99, 101 and 102, if not conserved between donor andacceptor, additionally comprise donor residues. Most preferably thelight chain framework additionally comprises donor residues at positions2, 4, 6, 35, 36, 38, 44, 47, 49, 62, 64-69, 85, 87, 98, 99, 101 and 102.

In addition the framework of the second or third aspects optionallycomprises donor residues at one, some or all of positions:

1 and 3,

63,

60 (if 60 and 54 are able to form at potential saltbridge),

70 (if 70 and 24 are able to form a potential saltbridge),

73 and 21 (if 47 is different between donor and acceptor),

37 and 45 (if 47 is different between donor and acceptor), and

any one or more of 10, 12, 40, 80, 103 and 105.

Preferably, the antigen binding regions of the CDR-grafted light chainvariable domain comprise CDRs corresponding to the Kabat CDRs at CDR1(residue 24-34), CDR2 (residues 50-56) and CDR3 (residues 89-97).

The invention further provides in a fourth aspect a CDR-grafted antibodymolecule comprising at least one CDR-grafted heavy chain and at leastone CDR-grafted light chain according to the first and second or firstand third aspects of the invention.

The humanised antibody molecules and chains of the present invention maycomprise: a complete antibody molecule, having full length heavy andlight chains; a fragment thereof, such as a Fab, (Fab')₂ or FV fragment;a light chain or heavy chain monomer or dimer; or a single chainantibody, e.g. a single chain FV in which heavy and light chain variableregions are joined by a peptide linker; or any other CDR-graftedmolecule with the same specificity as the original donor antibody.Similarly the CDR-grafted heavy and light chain variable region may becombined with other antibody domains as appropriate.

Also the heavy or light chains or humanised antibody molecules of thepresent invention may have attached to them an effector or reportermolecule. For instance, it may have a macrocycle, for chelating a heavymetal atom, or a toxin, such as ricin, attached to it by a covalentbridging structure. Alternatively, the procedures of recombinant DNAtechnology may be used to produce an immunoglobulin molecule in whichthe Fc fragment or CH3 domain of a complete immunoglobulin molecule hasbeen replaced by, or has attached thereto by peptide linkage, afunctional non-immunoglobulin protein, such as an enzyme or toxinmolecule.

Any appropriate acceptor variable region framework sequences may be usedhaving regard to class-type of the donor antibody from which the antigenbinding regions are derived. Preferably, the type of acceptor frameworkused is of the same/similar class/type as the donor antibody.Conveniently, the framework may be chosen to maximise/optimise homologywith the donor antibody sequence particularly at positions close oradjacent to the CDRs. However, a high level of homology between donorand acceptor sequences is not important for application of the presentinvention. The present invention identifies a hierarchy of frameworkresidue positions at which donor residues may be important or desirablefor obtaining a CDR-grafted antibody product having satisfactory bindingproperties. The CDR-grafted products usually have binding affinities ofat least 10⁵ M⁻¹, preferably at least about 10⁸ M⁻¹, or especially inthe range 10⁸-10¹² M⁻¹. In principle, the present invention isapplicable to any combination of donor and acceptor antibodiesirrespective of the level of homology between their sequences. Aprotocol for applying the invention to any particular donor-acceptorantibody pair is given hereinafter. Examples of human frameworks whichmay be used are KOL, NEWM, REI, EU, LAY and POM (refs. 4 and 5) and thelike; for instance KOL and NEWM for the heavy chain and REI for thelight chain and EU, LAY and POM for both the heavy chain and the lightchain.

Also the constant region domains of the products of the invention may beselected having regard to the proposed function of the antibody inparticular the effector functions which may be required. For example,the constant region domains may be human IgA, IgE, IgG or IgM domains.In particular, IgG human constant region domains may be used, especiallyof the IgG1 and IgG3 isotypes, when the humanised antibody molecule isintended for therapeutic uses, and antibody effector functions arerequired. Alternatively, IgG2 and IgG4 isotypes may be used when thehumanised antibody molecule is intended for therapeutic purposes andantibody effector functions are not required, e.g. for simple blockingof lymphokine activity.

However, the remainder of the antibody molecules need not comprise onlyprotein sequences from immunoglobulins. For instance, a gene may beconstructed in which a DNA sequence encoding part of a humanimmunoglobulin chain is fused to a DNA sequence encoding the amino acidsequence of a functional polypeptide such as an effector or reportermolecule.

Preferably the CDR-grafted antibody heavy and light chain and antibodymolecule products are produced by recombinant DNA technology.

Thus in further aspects the invention also includes DNA sequences codingfor the CDR-grafted heavy and light chains, cloning and expressionvectors containing the DNA sequences, host cells transformed with theDNA sequences and processes for producing the CDR-grafted chains andantibody molecules comprising expressing the DNA sequences in thetransformed host cells.

The general methods by which the vectors may be constructed,transfection methods and culture methods are well known per se and formno part of the invention. Such methods are shown, for instance, inreferences 10 and 11.

The DNA sequences which encode the donor amino acid sequence may beobtained by methods well known in the art. For example the donor codingsequences may be obtained by genomic cloning, or cDNA cloning fromsuitable hybridoma cell lines. Positive clones may be screened usingappropriate probes for the heavy and light chain genes in question. AlsoPCR cloning may be used.

DNA coding for acceptor, e.g. human acceptor, sequences may be obtainedin any appropriate way. For example DNA sequences coding for preferredhuman acceptor frameworks such as KOL, REI, EU and NEWM, are widelyavailable to workers in the art.

The standard techniques of molecular biology may be used to prepare DNAsequences coding for the CDR-grafted products. Desired DNA sequences maybe synthesised completely or in part using oligonucleotide synthesistechniques. Site--directed mutagenesis and polymerase chain reaction(PCR) techniques may be used as appropriate. For example oligonucleotidedirected synthesis as described by Jones et al (ref 20) may be used.Also oligonucleotide directed mutagenesis of a pre-existing variableregion as, for example, described by Verhoeyen et al (ref. 5) orRiechmann et al (ref. 6) may be used. Also enzymatic filling in ofgapped oligonucleotides using T₄ DNA polymerase as, for example,described by Queen et al (ref. 9) may be used.

Any suitable host cell/vector system may be used for expression of theDNA sequences coding for the CDR-grafted heavy and light chains.Bacterial e.g. E. coli, and other microbial systems may be used, inparticular for expression of antibody fragments such as FAb and (Fab')₂fragments, and especially FV fragments and single chain antibodyfragments e.g. single chain FVs. Eucaryotic e.g. mammalian host cellexpression systems may be used for production of larger CDR-graftedantibody products, including complete antibody molecules. Suitablemammalian host cells include CHO cells and myeloma or hybridoma celllines.

Thus, in a further aspect the present invention provides a process forproducing a CDR-grafted antibody product comprising:

-   (a) producing in an expression vector an operon having a DNA    sequence which encodes an antibody heavy chain according to the    first aspect of the invention;    and/or-   (b) producing in an expression vector an operon having a DNA    sequence which encodes a complementary antibody light chain    according to the second or third aspect of the invention;-   (c) transfecting a host cell with the or each vector; and-   (d) culturing the transfected cell line to produce the CDR-grafted    antibody product.

The CDR-grafted product may comprise only heavy or light chain derivedpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence is used to transfect the host cells. For production ofproducts comprising both heavy and light chains, the cell line may betransfected with two vectors, the first vector may contain an operonencoding a light chain-derived polypeptide and the second vectorcontaining an operon encoding a heavy chain-derived polypeptide.Preferably, the vectors are identical, except in so far as the codingsequences and selectable markers are concerned, so as to ensure as faras possible that each polypeptide chain is equally expressed.Alternatively, a single vector may be used, the vector including thesequences encoding both light chain- and heavy chain-derivedpolypeptides.

The DNA in the coding sequences for the light and heavy chains maycomprise cDNA or genomic DNA or both. However, it is preferred that theDNA sequence encoding the heavy or light chain comprises at leastpartially, genomic DNA, preferably a fusion of cDNA and genomic DNA.

The present invention is applicable to antibodies of any appropriatespecificity. Advantageously, however, the invention may be applied tothe humanisation of non-human antibodies which are used for in vivotherapy or diagnosis. Thus the antibodies may be site-specificantibodies such as tumour-specific or cell surface-specific antibodies,suitable for use in in vivo therapy or diagnosis, e.g. tumour imaging.Examples of cell surface-specific antibodies are anti-T cell antibodies,such as anti-CD3, and CD4 and adhesion molecules, such as CR3, ICAM andELAM. The antibodies may have specificity for interleukins (includinglymphokines, growth factors and stimulating factors), hormones and otherbiologically active compounds, and receptors for any of these. Forexample, the antibodies may have specificity for any of the following:Interferons α, β, γ or δ, IL1, IL2, IL3, or IL4, etc., TNF, GCSF, GMCSF,EPO, hGH, or insulin, etc.

The the present invention also includes therapeutic and diagnosticcompositions comprising the CDR-grafted products of the invention anduses of such compositions in therapy and diagnosis.

Accordingly in a further aspect the invention provides a therapeutic ordiagnostic composition comprising a CDR-grafted antibody heavy or lightchain or molecule according to previous aspects of the invention incombination with a pharmaceutically acceptable carrier, diluent orexcipient.

Accordingly also the invention provides a method of therapy or diagnosiscomprising administering an effective amount of a CDR-grafted antibodyheavy or light chain or molecule according to previous aspects of theinvention to a human or animal subject.

A preferred protocol for obtaining CDR-grafted antibody heavy and lightchains in accordance with the present invention is set out belowtogether with the rationale by which we have derived this protocol. Thisprotocol and rationale are given without prejudice to the generality ofthe invention as hereinbefore described and defined.

Protocol

It is first of all necessary to sequence the DNA coding for the heavyand light chain variable regions of the donor antibody, to determinetheir amino acid sequences. It is also necessary to choose appropriateacceptor heavy and light chain variable regions, of known amino acidsequence. The CDR-grafted chain is then designed starting from the basisof the acceptor sequence. It will be appreciated that in some cases thedonor and acceptor amino acid residues may be identical at a particularposition and thus no change of acceptor framework residue is required.

-   1. As a first step donor residues are substituted for acceptor    residues in the CDRs. For this purpose the CDRs are preferably    defined as follows:

Heavy chain CDR1: residues 26-35 CDR2: residues 50-65 CDR3: residues95-102 Light chain CDR1: residues 24-34 CDR2: residues 50-56 CDR3:residues 89-97

-    The positions at which donor residues are to be substituted for    acceptor in the framework are then chosen as follows, first of all    with respect to the heavy chain and subsequently with respect to the    light chain.-   2. Heavy Chain-   2.1 Choose donor residues at all of positions 23, 24, 49, 71, 73 and    78 of the heavy chain or all of positions 23, 24 and 49 (71, 73 and    78 are always either all donor or all acceptor).-   2.2 Check that the following have the same amino acid in donor and    acceptor sequences, and if not preferably choose the donor: 2, 4, 6,    25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.-   2.3 To further optimise affinity consider choosing donor residues at    one, some or any of:    -   i. 1, 3    -   ii. 72, 76    -   iii. If 48 is different between donor and acceptor sequences,        consider 69    -   iv. If at 48 the donor residue is chosen, consider 38 and 46    -   v. If at 69 the donor residue is chosen, consider 80 and then 20    -   vi. 67    -   vii. If at 67 the donor residue is chosen, consider 82 and then        18    -   viii. 91    -   ix. 88    -   x. 9, 11, 41, 87, 108, 110, 112-   3. Light Chain-   3.1 Choose donor at 46, 48, 58 and 71-   3.2 Check that the following have the same amino acid in donor and    acceptor sequences, if not preferably choose donor:    -   2, 4, 6, 35, 38, 44, 47, 49, 62, 64-69 inclusive, 85, 87, 98,        99, 101 and 102-   3.3 To further optimise affinity consider choosing donor residues at    one, some or any of:    -   i. 1,3    -   ii. 63    -   iii. 60, if 60 and 54 are able to form potential saltbridge    -   iv. 70 and 24 are able to form potential saltbridge    -   v. 73, and 21 if 47 is different between donor and acceptor    -   vi. 37, and 45 if 47 is different between donor and acceptor    -   vii. 10, 12, 40, 80, 103, 105        Rationale

In order to transfer the binding site of an antibody into a differentacceptor framework, a number of factors need to be considered.

1. The Extent of the CDRs

The CDRs (Complementary Determining Regions) were defined by Wu andKabat (refs. 4 and 5) on the basis of an analysis of the variability ofdifferent regions of antibody variable regions. Three regions per domainwere recognised. In the light chain the sequences are 24-24, 50-56,89-97 (numbering according to Kabat (ref 4), Eu Index) inclusive and inthe heavy chain the sequences are 31-35, 50-65 and 95-102 inclusive.

When antibody structures became available it became apparent that theseCDR regions corresponded in the main to loop regions which extended fromthe β barrel framework of the light and heavy variable domains. For H1there was a discrepancy in that the loop was from 26 to 32 inclusive andfor H2 the loop was 52 to 56 and for L2 from 50 to 53. However, with theexception of H1 the CDR regions encompassed the loop regions andextended into the β strand frameworks. In H1 residue 26 tends to be aserine and 27 a phenylalanine or tyrosine, residue 29 is a phenylalaninein most cases. Residues 28 and 30 which are surface residues exposed tosolvent might be involved in antigen-binding. A prudent definition ofthe H1 CDR therefore would include residues 26-35 to include both theloop region and the hypervariable residues 33-35.

It is of interest to note the example of Riechmann et al (ref. 3), whoused the residue 31-35 choice for CDR-H1. In order to produce efficientantigen binding, residue 27 also needed to be recruited from the donor(rat) antibody.

2. Non-CDR Residues Which Contribute to Antigen Binding

By examination of available X-ray structures we have identified a numberof residues which may have an effect on net antigen binding and whichcan be demonstrated by experiment. These residues can be sub-dividedinto a number of groups.

-   2.1 Surface residues near CDR [all numbering as in Kabat et al (ref.    7)].-   2.1.1. Heavy Chain—Key residues are 23, 71 and 73. Other residues    which may contribute to a lesser extent are 1, 3 and 76. Finally 25    is usually conserved but the murine residue should be used if there    is a difference.-   2.1.2 Light Chain—Many residues close to the CDRs, e.g. 63, 65, 67    and 69 are conserved. If conserved none of the surface residues in    the light chain are likely to have a major effect. However, if the    murine residue at these positions is unusual, then it would be of    benefit to analyse the likely contribution more closely. Other    residues which may also contribute to binding are 1 and 3, and also    60 and 70 if the residues at these positions and at 54 and 24    respectively are potentially able to form a salt bridge i.e. 60+54;    70+24.-   2.2 Packing residues near the CDRs.-   2.2.1. Heavy Chain—Key residues are 24, 49 and 78. Other key    residues would be 36 if not a tryptophan, 94 if not an arginine, 104    and 106 if not glycines and 107 if not a threonine. Residues which    may make a further contribution to stable packing of the heavy chain    and hence improved affinity are 2, 4, 6, 38, 46, 67 and 69. 67 packs    against the CDR residue 63 and this pair could be either both mouse    or both human. Finally, residues which contribute to packing in this    region but from a longer range are 18, 20, 80, 82 and 86. 82 packs    against 67 and in turn 18 packs against 82. 80 packs against 69 and    in turn 20 packs against 80. 86 forms an H bond network with 38    and 46. Many of the mouse-human differences appear minor e.g.    Leu-Ile, but could have an minor impact on correct packing which    could translate into altered positioning of the CDRs.-   2.2.2. Light Chain—Key residues are 48, 58 and 71. Other key    residues would be 6 if not glutamine, 35 if not tryptophan, 62 if    not phenylalanine or tryosine, 64, 66, 68, 99 and 1010 if not    glycines and 102 if not a threonine. Residues which make a further    contribution are 2, 4, 37, 45 and 47. Finally residues 73 and 21 and    19 may make long distance packing contributions of a minor nature.-   2.3. Residues at the variable domain interface between heavy and    light chains—In both the light and heavy chains most of the non-CDR    interface residues are conserved. If a conserved residue is replaced    by a residue of different character, e.g. size or charge, it should    be considered for retention as the murine residue.-   2.3.1. Heavy Chain—Residues which need to be considered are 37 if    the residue is not a valine but is of larger side chain volume or    has a charge or polarity. Other residues are 39 if not a glutamine,    45 if not a leucine, 47 if not a tryptophan, 91 if not a    phenylalanine or tyrosine, 93 if not an alanine and 103 if not a    tryptophan. Residue 89 is also at the interface but is not in a    position where the side chain could be of great impact.-   2.3.2. Light Chain—Residues which need to be considered are 36, if    not a tyrosine, 38 if not a glutamine, 44 if not a proline, 46, 49    if not a tyrosine, residue 85, residue 87 if not a tyrosine and 98    if not a phenylalanine.-   2.4. Variable-Constant region interface—The elbow angle between    variable and constant regions may be affected by alterations in    packing of key residues in the variable region against the constant    region which may affect the position of V_(L) and V_(H) with respect    to one another. Therefore it is worth noting the residues likely to    be in contact with the constant region. In the heavy chain the    surface residues potentially in contact with the variable region are    conserved between mouse and human antibodies therefore the variable    region contact residues may influence the V-C interaction. In the    light chain the amino acids found at a number of the constant region    contact points vary, and the V & C regions are not in such close    proximity as the heavy chain. Therefore the influences of the light    chain V-C interface may be minor.-   2.4.1. Heavy Chain—Contact residues are 7, 11, 41, 87, 108, 110,    112.-   2.4.2. Light Chain—In the light chain potentially contacting    residues are 10, 12, 40, 80, 83, 103 and 105.

The above analysis coupled with our considerable practical experimentalexperience in the CDR-grafting of a number of different antibodies havelead us to the protocol given above.

The present invention is now described, by way of example only, withreference to the accompanying FIGS. 1-13.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1a and 1b show DNA and amino acid sequences of the OKT3 lightchain (SEQ ID NO: 4 and 5);

FIGS. 2a and b show DNA and amino acid sequences of the OKT3 heavy chain(SEQ ID NO: 6 and 7);

FIG. 3 shows the alignment of the OKT3 light variable region amino acidsequence with that of the light variable region of the human antibodyREI (SEQ ID NO: 5 and 8);

FIG. 4 shows the alignment of the OKT3 heavy variable region amino acidsequence with that of the heavy variable region of the human antibodyKOL (SEQ ID NO: 7 and 10);

FIGS. 5a-c show the heavy variable region amino acid sequences of OKT3,KOL and various corresponding CDR grafts (SEQ ID NO: 7 and 11-24);

FIG. 6 shows the light variable region amino acid sequences of OKT3, REIand various corresponding CDR grafts (SEQ ID NO: 5, 8, 9, and 25-28);

FIG. 7 shows a graph of binding assay results for various grafted OKT3antibodies'

FIG. 8 shows a graph of blocking assay results for various grafted OKT3antibodies;

FIG. 9 shows a similar graph of blocking assay results;

FIGS. 10a and b show similar graphs for both binding assay and blockingassay results;

FIGS. 11a and b show further similar graphs for both binding assay andblocking assay results;

FIG. 12 shows a graph of competition assay results for a minimallygrafted OKT3 antibody compared with the OKT3 murine reference standard,and

FIG. 13 shows a similar graph of competition assay results comparing afully grafted OKT3 antibody with the murine reference standard.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION Example 1

CDR-Grafting of OKT3

Material and Methods

1. Incoming Cells

Hybridoma cells producing antibody OKT3 were provided by Ortho (seedlot4882.1) and were grown up in antibiotic free Dulbecco's Modified EaglesMedium (DMEM) supplemented with glutamine and 5% foetal calf serum, anddivided to provide both an overgrown supernatant for evaluation andcells for extraction of RNA. The overgrown supernatant was shown tocontain 250 ug/mL murine IgG2a/kappa antibody. The supernatant wasnegative for murine lambda light chain and IgG1, IgG2b, IgG3, IgA andIgM heavy chain. 20 mL of supernatant was assayed to confirm that theantibody present was OKT3.

2. Molecular Biology Procedures

Basic molecular biology procedures were as described in Maniatis et al(ref 9) with, in some cases, minor modifications. DNA sequencing wasperformed as described in Sanger et al (ref. 11) and the AmershamInternational Plc sequencing handbook. Site directed mutagenesis was asdescribed in Kramer et al (ref 12) and the Anglian Biotechnology Ltd.handbook. COS cell expression and metabolic labelling studies were asdescribed in Whittle et al (ref. 13)

3. Research Assays

3.1. Assembly Assays

Assembly assays were performed on supernatants from transfected COScells to determine the amount of intact IgG present.

3.1.1. COS Cells Transfected with Mouse OKT3 Genes

The assembly assay for intact mouse IgG in COS cell supernatants was anELISA with the following format:

96 well microtiter plates were coated with F(ab')1 goat anti-mouse IgGFc. The plates were washed in water and samples added for 1 hour at roomtemperature. The plates were washed and F(ab')2 goat anti-mouse IgGF(ab')2 (HRPO conjugated) was then added. Substrate was added to revealthe reaction. UPC10, a mouse IgG2a myeloma, was used as a standard.

3.1.2. COS and CHO Cells Transfected with Chimeric or CDR-Grafted OKT3Genes

The assembly assay for chimeric or CDR-grafted antibody in COS cellsupernatants was an ELISA with the following format:

96 well microtiter plates were coated with F(ab')2 goat anti-human IgGFc. The plates were washed and samples added and incubated for 1 hour atroom temperature. The plates were washed and monoclonal mouse anti-humankappa chain was added for 1 hour at room temperature. The plates werewashed and F(ab')2 goat anti-mouse IgG Fc (HRPO conjugated) was added.Enzyme substrate was added to reveal the reaction. Chimeric B72.3 (IgG4)(ref. 13) was used as a standard. The use of a monoclonal anti-kappachain in this assay allows grafted antibodies to be read from thechimeric standard.

3.2. Assay for Antigen Binding Activity

Material from COS cell supernatants was assayed for OKT3 antigen bindingactivity onto CD3 positive cells in a direct assay. The procedure was asfollows:

HUT 78 cells (human T cell line, CD3 positive) were maintained inculture. Monolayers of HUT 78 cells were prepared onto 96 well ELISAplates using poly-L-lysine and glutaraldehyde. Samples were added to themonolayers for 1 hour at room temperature.

The plates were washed gently using PBS. F(ab')2 goat anti-human IgG Fc(HRPO conjugated) or F(ab')2 goat antimouse IgG Fc (HRPO conjugated) wasadded as appropriate for humanised or mouse samples. Substrate was addedto reveal the reaction. The negative control for the cell-based assaywas chimeric B72.3. The positive control was mouse Orthomune OKT3 orchimeric OKT3, when available. This cell-based assay was difficult toperform, and an alternative assay was developed for CDR-grafted OKT3which was more sensitive and easier to carry out.

In this system CDR-grafted OKT3 produced by COS cells was tested for itsability to bind to the CD3-positive HPB-ALL (human peripheral bloodacute lymphocytic leukemia) cell line. It was also tested for itsability to block the binding of murine OKT3 to these cells. Binding wasmeasured by the following procedure: HPB-ALL cells were harvested fromtissue culture. Cells were incubated at 4° C. for 1 hour with variousdilutions of test antibody, positive control antibody, or negativecontrol antibody. The cells were washed once and incubated at 4° C. for1 hour with an FITC-labelled goat anti-human IgG (Fc-specific, mouseabsorbed). The cells were washed twice and analysed by cytofluorography.Chimeric OKT3 was used as a positive control for direct binding. Cellsincubated with mock-transfected COS cell supernatant, followed by theFITC-labelled goat anti-human IgG, provided the negative control. Totest the ability of CDR-grafted OKT3 to block murine OKT3 binding, theHPB-ALL cells were incubated at 4° C. for 1 hour with various dilutionsof test antibody or control antibody. A fixed saturating amount of FITCOKT3 was added. The samples were incubated for 1 hour at 4° C., washedtwice and analysed by cytofluorography. FITC-labelled OKT3 was used as apositive control to determine maximum binding. Unlabelled murine OKT3served as a reference standard for blocking. Negative controls wereunstained cells with or without mock-transfected cell supernatant. Theability of the CDR-grafted OKT3 light chain to bind CD3-positive cellsand block the binding of murine OKT3 was initially tested in combinationwith the chimeric OKT3 heave chain. The chimeric OKT3 heavy chain iscomposed of the murine OKT3 variable region and the human IgG4 constantregion. The chimeric heavy chain gene is expressed in the sameexpression vector used for the CDR-grafted genes. The CDR-grafted lightchain expression vector and the chimeric heavy chain expression vectorwere co-transfected into COS cells. The fully chimeric OKT3 antibody(chimeric light chain and chimeric heavy chain) was found to be fullycapable of binding to CD3 positive cells and blocking the binding ofmurine OKT3 to these cells.

3.3 Determination of Relative Binding Affinity

The relative binding affinities of CDR-grafted anti-CD3 monoclonalantibodies were determined by competition binding (ref. 6) using theHPB-ALL human T cell line as a source of CD3 antigen, andfluorescein-conjugated murine OKT3 (Fl-OKT3) of known binding affinityas a tracer antibody. The binding affinity of Fl-OKT3 tracer antibodywas determined by a direct binding assay in which increasing amounts ofFl-OKT3 were incubated with HPB-ALL (5×10⁵) in PBS with 5% foetal calfserum for 60 min. at 4° C. Cells were washed, and the fluorescenceintensity was determined on a FACScan flow cytometer calibrated withquantitative microbead standards (Flow Cytometry Standards, ResearchTriangle Park, N.C.). Fluorescence intensity per antibody molecule (F/Pratio) was determined by using microbeads which have a predeterminednumber of mouse IgG antibody binding sites (Simply Cellular beads, FlowCytometry Standards). F/P equals the fluorescence intensity of beadssaturated with Fl-OKT3 divided by the number of binding sites per bead.The amount of bound and free Fl-OKT3 was calculated from the meanfluorescence intensity per cell, and the ratio of bound/free was plottedagainst the number of moles of antibody bound. A linear fit was used todetermine the affinity of binding (absolute value of the slope).

For competitive binding, increasing amounts of competitor antibody wereadded to a sub-saturating dose of Fl-OKT3 and incubated with 5×10⁵HPB-ALL in 200 ml of PBS with 5% foetal calf serum, for 60 min at 4° C.The fluorescence intensities of the cells were measured on a FACScanflow cytometer calibrated with quantitative microbead standards. Theconcentrations of bound and free Fl-OKT3 were calculated. The affinitiesof competing antibodies were calculated from the equation[X]−[OKT3]−(1/Kx)−(1/Ka), where Ka is the affinity of murine OKT3, Kx isthe affinity of competitor X, [ ] is the concentration of competitorantibody at which bound/free binding is R/2, and R is the maximalbound/free binding.

4. cDNA Library Construction

4.1 mRNA Preparation and cDNA Synthesis

OKT3 producing cells were grown as described above and 1.2×10⁹ cellsharvested and mRNA extracted using the guanidinium/LiC1 extractionprocedure. cDNA was prepared by priming from Oligo-dT to generate fulllength cDNA. The cDNA was methylated and EcoR1 linkers added forcloning.

4.2. Library Construction

The cDNA library was ligated to pSP65 vector DNA which had been EcoR1cut and the 5′ phosphate groups removed by calf intestinal phosphatase(EcoR1/CIP). The ligation was used to transform high transformationefficiency Escherichia coli (E. coli) HB101. A cDNA library wasprepared. 3600 colonies were screened for the light chain and 10000colonies were screened for the heavy chain.

5. Screening

E. coli colonies positive for either heavy or light chain probes wereidentified by oligonucleotide screening using the oligonucleotides: 5′TCCAGATGTTAACTGCTCAC (SEQ ID NO: 1) for the light chain, which iscomplementary to a sequence in the mouse kappa constant region, and 5′CAGGGGCCAGTGGATGGATAGAC (SEQ ID NO: 2) for the heavy chain which iscomplementary to a sequence in the mouse IgG2a constant CH1 domainregion. 12 light chain and 9 heavy chain clones were identified andtaken for second round screening. Positive clones from the second roundof screening were grown up and DNA prepared. The sizes of the geneinserts were estimated by gel electrophoresis and inserts of a sizecapable of containing a full length cDNA were subcloned into M13 for DNAsequencing.

6. DNA Sequencing

Clones representing four size classes for both heavy and light chainswere obtained in M13. DNA sequence for the 5′ untranslated regions,signal sequences, variable regions and 3′ untranslated regions of fulllength cDNAs [FIGS. 1(a) and 2(a)] were obtained and the correspondingamino acid sequences predicted [(FIGS. 1(b) and 2(b)]. In FIG. 1(a) theuntranslated DNA regions are shown in uppercase, and in both FIGS. 1 and2 the signal sequences are underlined.

7. Construction of cDNA Expression Vectors

Celltech expression vectors are based on the plasmid pEE6hCMV (ref 14).A polylinker for the insertion of genes to be expressed has beenintroduced after the major immediate early promoter/enhancer of thehuman Cytomegalovirus (hCMV). Marker genes for selection of the plasmidin transfected eukaryotic cells can be inserted as BamH1 cassettes inthe unique BamH1 site of pEE6 hCMV; for instance, the neo marker toprovide pEE6 hCMV neo. It is usual practice to insert the neo and gptmarkers prior to insertion of the gene of interest, whereas the GSmarker is inserted last because of the presence of internal EcoR1 sitesin the cassette.

The selectable markers are expressed from the SV40 late promoter whichalso provides an origin of replication so that the vectors can be usedfor expression in the COS cell transient expression system.

The mouse sequences were excised from the M13 based vectors describedabove as EcoR1 fragments and cloned into either pEE6-hCMV-neo for theheavy chain and into EE6hCMV-gpt for the light chain to yield vectorspJA136 and pJA135 respectively.

8. Expression of cDNAs in COS Cells.

Plasmids pJA135 and pJA136 were co-transfected into COS cells andsupernatant from the transient expression experiment was shown tocontain assembled antibody which bound to T-cell enriched lymphocytes.Metabolic labelling experiments using ³⁵S methionine showed expressionand assembly of heavy and light chains.

9. Construction of Chimeric Genes

Construction of chimeric genes followed a previously described strategy[Whittle et al (ref. 13)]. A restriction site near the 3′ end of thevariable domain sequence is identified and used to attach anoligonucleotide adapter coding for the remainder of the mouse variableregion and a suitable restriction site for attachment to the constantregion of choice.

9.1. Light Chain Gene Construction

The mouse light chain cDNA sequence contains an Ava1 site near the 3′end of the variable region [FIG. 1(a)]. The majority of the sequence ofthe variable region was isolated as a 396 bp. EcoR1-Ava1 fragment. Anoligonucleotide adapter was designed to replace the remainder of the 3′region of the variable region from the Ava1 site and to include the 5′residues of the human constant region up to and including a unique Nar1site which had been previously engineered into the constant region.

A Hind111 site was introduced to act as a marker for insertion of thelinker.

The linker was ligated to the V_(L) fragment and the 413 bp EcoR1-Nar1adapted fragment was purified from the ligation mixture.

The constant region was isolated as an Nar1 -BamH1 fragment from an M13clone NW361 and was ligated with the variable region DNA into anEcoR1/BamH1/C1P pSP65 treated vector in a three way reaction to yieldplasmid JA143. Clones were isolated after transformation into E. coliand the linker and junction sequences were confirmed by the presence ofthe Hind111 site and by DNA sequencing.

9.2 Light Chain Gene Construction—Version 2

The construction of the first chimeric light chain gene produces afusion of mouse and human amino acid sequences at the variable-constantregion junction. In the case of the OKT3 light chain the amino acids atthe chimera junction are:...Leu-Glu-Ile-Asn-Arg(SEQ ID NO:3)/-/Thr-Val-Ala-Ala

(SEQ ID NO: 3) Leu-Glu-Ile-Asn-Arg/   -/Thr-Val-Ala  -Ala            VARIABLE         CONSTANTThis arrangement of sequence introduces a potential site for Asparagine(Asn) linked (N-linked) glycosylation at the V-C junction. Therefore, asecond version of the chimeric light chain oligonucleotide adapter wasdesigned in which the threonine (Thr), the first amino acid of the humanconstant region, was replaced with the equivalent amino acid from themouse constant region, Alanine (Ala).

An internal Hind111 site was not included in this adapter, todifferentiate the two chimeric light chain genes.

The variable region fragment was isolated as a 376 bp EcoR1-Ava1fragment. The oligonucleotide linker was ligated to Nar1 cut pNW361 andthen the adapted 396 bp constant region was isolated after recutting themodified pNW361 with EcoR1. The variable region fragment and themodified constant region fragment were ligated directly into EcoR1/C1Ptreated pEE6hCMVneo to yield pJA137. Initially all clones examined hadthe insert in the incorrect orientation. Therefore, the insert wasre-isolated and recloned to turn the insert round and yield plasmidpJA141. Several clones with the insert in the correct orientation wereobtained and the adapter sequence of one was confirmed by DNA sequencing

9.3. Heavy Chain Gene Construction

9.3.1. Choice of Heavy Chain Gene Isotype

The constant region isotype chosen for the heavy chain was human IgG4.

9.3.2. Gene Construction

The heavy chain cDNA sequence showed a Ban1 site near the 3′ end of thevariable region [FIG. 2(a)]. The majority of the sequence of thevariable region was isolated as a 426 bp. EcoR1/C1P/Ban1 fragment. Anoligonucleotide adapter was designated to replace the remainder of the3′ region of the variable region from the Ban1 site up to and includinga unique HindIII site which had been previously engineered into thefirst two amino acids of the constant region. The linker was ligated tothe V_(H) fragment and the EcoR1-Hind111 adapted fragment was purifiedfrom the ligation mixture.

The variable region was ligated to the constant region by cutting pJA91with EcoR1 and Hind111 removing the intron fragment and replacing itwith the V_(H) to yield pJA142. Clones were isolated aftertransformation in E. coli JM101 and the linker and junction sequenceswere confirmed by DNA sequencing. (N.B. The Hind111 site is lost oncloning).

10. Construction of Chimeric Expression Vectors

10.1. neo AND gpt Vectors

The chimeric light chain (version 1) was removed from pJA143 as an EcoR1fragment and cloned into EcoR1/C1P treated pEE6hCMVneo expression vectorto yield pJA145. Clones with the insert in the correct orientation wereidentifled by restriction mapping.

The chimeric light chain (version 2) was constructed as described above.

The chimeric heavy chain gene was isolated from pJA142 as a 2.5 KbpEcoR1/BamH1 fragment and cloned into the EcoR1/Bc11/C1P treated vectorfragment of a derivative of pEE6hCMVgpt to yield plasmid pJA144.

10.2. GS Separate Vectors

GS versions of pJA141 and pJA144 were constructed by replacing the neoand gpt cassettes by a BamH1/Sa11/C1P treatment of the plasmids,isolation of the vector fragment and ligation to a GS-containingfragment from the plasmid pRO49 to yield the light chain vector pJA179and the heavy chain vector pJA180.

10.3. GS Single Vector Construction

Single vector constructions containing the cL (chimeric light), cH(chimeric heavy) and GS genes on one plasmid in the order cL-cH-GS, orcH-cL-GS and with transcription of the genes being head to tail e.g.cL>cH>GS were constructed. These plasmids were made by treating pJA179or pJA180 with BamH1/C1P and ligating in a Bg111/Hind111 hCMV promotercassette along with either the Hind111/BamH1 fragment from pJA141 intopJA180 to give the cH-cL-GS plasmid pJA182 or the Hind111/BamH1 fragmentfrom pJA144 into pJA179 to give the cL-cH-GS plasmid pJA181.

11. Expression of Chimeric Genes

11.1. Expression in COS Cells

The chimeric antibody plasmid pJA145 (cL) and pJA144 (cH) wereco-transfected into COS cells and supernatant from the transientexpression experiment was shown to contain assembled antibody whichbound to the HUT 78 human T-cell line. Metabolic labelling experimentsusing ³⁵S methionine showed expression and assembly of heavy and lightchains. However the light chain mobility seen on reduced gels suggestedthat the potential glycosylation site was being glycosylated. Expressionin COS cells in the presence of tunicamycin showed a reduction in sizeof the light chain to that shown for control chimeric antibodies and theOKT3 mouse light chain. Therefore JA141 was constructed and expressed.In this case the light chain did not show an aberrant mobility or a sizeshift in the presence or absence of tunicamycin. This second version ofthe chimeric light chain, when expressed in association with chimericheavy (cH) chain, produced antibody which showed good binding to HUT 78cells. In both cases antigen binding was equivalent to that of the mouseantibody.

11.2 Expression in Chinese Hamster Ovary (CHO) Cells

Stable cell lines have been prepared from plasmids pJA141/pJA144 andfrom pJA179/pJA180, pJA181 and pJA182 by transfection into CHO cells.

12. CDR-Grafting

The approach taken was to try to introduce sufficient mouse residuesinto a human variable region framework to generate antigen bindingactivity comparable to the mouse and chimeric antibodies.

12.1. Variable Region Analysis

From an examination of a small database of structures of antibodies andantigen-antibody complexes it is clear that only a small number ofantibody residues make direct contact with antigen. Other residues maycontribute to antigen binding by positioning the contact residues infavourable configurations and also by inducing a stable packing of theindividual variable domains and stable interaction of the light andheavy chain variable domains. The residues chosen for transfer can beidentified in a number of ways:

-   -   (a) By examination of antibody X-ray crystal structures the        antigen binding surface can be predominantly located on a series        of loops, three per domain, which extend from the B-barrel        framework.    -   (b) By analysis of antibody variable domain sequences regions of        hypervariability [termed the Complementarity Determining Regions        (CDRs) by Wu and Kabat (ref. 5)] can be identified. In the most        but not all cases these CDRs correspond to, but extend a short        way beyond, the loop regions noted above.    -   (c) Residues not identified by (a) and (b) may contribute to        antigen binding directly or indirectly by affecting antigen        binding site topology, or by inducing a stable packing of the        individual variable domains and stabilising the inter-variable        domain interaction. These residues may be identified either by        superimposing the sequences for a given antibody on a known        structure and looking at key residues for their contribution, or        by sequence alignment analysis and noting “idiosyncratic”        residues followed by examination of their structural location        and likely effects.        12.1.1. Light Chain

FIG. 3 shows an alignment of sequences for the human framework regionRE1 and the OKT3 light variable region. The structural loops (LOOP) andCDRs (KABAT) believed to correspond to the antigen binding region aremarked. Also marked are a number of other residues which may alsocontribute to antigen binding as described in 13.1(c). Above thesequence in FIG. 3 the residue type indicates the spatial location ofeach residue side chain, derived by examination of resolved structuresfrom X-ray crystallography analysis. The key to this residue typedesignation is as follows:

N—near to CDR (From X-ray Structures) P—Packing B—Buried Non-PackingS—Surface E—Exposed I—Interface *—Interface  —Packing/Part Exposed?—Non-CDR Residues which may require to be left as Mouse sequence.

Residues underlined in FIG. 3 are amino acids. RE1 was chosen as thehuman framework because the light chain is a kappa chain and the kappavariable regions show higher homology with the mouse sequences than alambda light variable region, e.g. KOL (see below). RE1 was chosen inpreference to another kappa light chain because the X-ray structure ofthe light chain has been determined so that a structural examination ofindividual residues could be made.

12.1.2. Heavy Chain

Similarly FIG. 4 shows an alignment of sequences for the human frameworkregion KOL and the OKT3 heavy variable region. The structural loops andCDRs believed to correspond to the antigen binding region are marked.Also marked are a number of other residues which may also contribute toantigen binding as described in 12.1(c). The residue type key and otherindicators used in FIG. 4 are the same as those used in FIG. 3. KOL waschosen as the heavy chain framework because the X-ray structure has beendetermined to a better resolution than, for example, NEWM and also thesequence alignment of OKT3 heavy variable region showed a slightlybetter homology to KOL than to NEWM.

12.2. Design of Variable Genes

The variable region domains were designed with mouse variable regionoptimal codon usage [Granthan and Perrin (ref 15)] and used the B72.3signal sequences [Whittle et al (ref 13)]. The sequences were designedto be attached to the constant region in the same way as for thechimeric genes described above. Some constructs contained the “Kozakconsensus sequence” [Kozak (ref 16)] directly linked to the 5′ of thesignal sequence in the gene. This sequence motif is believed to have abeneficial role in translation initiation in eukaryotes.

12.3. Gene Construction

To build the variable regions, various strategies are available. Thesequence may be assembled by using oligonucleotides in a manner similarto Jones et al (ref. 17) or by simultaneously replacing all of the CDRsor loop regions by oligonucleotide directed site specific mutagenesis ina manner similar to Verhoeyen et al (ref 2). Both strategies were usedand a list of constructions is set out in Tables 1 and 2 and FIGS. 4 and5a-c. It was noted in several cases that the mutagenesis approach led todeletions and rearrangements in the gene being remodelled, while thesuccess of the assembly approach was very sensitive to the quality ofthe oligonucleotides.

13. Construction of Expression Vectors

Genes were isolated from M13 or SP65 based intermediate vectors andcloned into pEE6hCMVneo for the light chains and pEE6hCMVgpt for theheavy chains in a manner similar to that for the chimeric genes asdescribed above.

TABLE 1 CDR-GRAFTED GENE CONSTRUCTS METHOD OF KOZAK SEQUENCE CODE MOUSESEQUENCE CONTENT CONSTRUCTION − + LIGHT CHAIN ALL HUMAN FRAMEWORK RE1121 26-32, 50-56, 91-96 inclusive SDM and gene assembly + n.d. 121A26-32, 50-56, 91-96 inclusive Partial gene assembly n.d. + +1, 3, 46, 47121B 26-32, 50-56, 91-96 inclusive Partial gene assembly n.d. + +46, 47221 24-24, 50-56, 91-96 inclusive Partial gene assembly + + 221A 24-34,50-56, 91-96 inclusive Partial gene assembly + + +1, 3, 46, 47 221B24-34, 50-56, 91-96 inclusive Partial gene assembly + + +1, 3 221C24-34, 50-56, 91-96 inclusive Partial gene assembly + + HEAVY CHAIN ALLHUMAN FRAMEWORK KOL 121 26-32, 50-56, 95-100B inclusive Gene assemblyn.d + 131 26-32, 50-58, 95-100B inclusive Gene assembly n.d. + 14126-32, 50-65, 95-100B inclusive Partial gene assembly + n.d. 321 26-35,50-56, 95-100B inclusive Partial gene assembly + n.d. 331 26-35, 50-58,95-100B inclusive Partial gene assembly + Gene assembly + 341 26-35,50-65, 95-100B inclusive SDM + Partial gene assembly + 341A 26-35,50-65, 95-100B inclusive Gene assembly n.d. + +6, 23, 24, 48, 49, 71,73, 76, 78, 88, 91 (+63 = human) 341B 26-35, 50-65, 95-100B inclusiveGene assembly n.d. + +48, 49, 71, 73, 76, 78, 88, 91 (+63 + human) (SEQID NO:8-28) KEY n.d. not done SDM Site directed mutagenesis Geneassembly Variable region assembled entirely from oligonucleotidesPartial gene assembly Variable region assembled by combination ofrestriction fragments either from other genes originally created by SDMand gene assembly or by oligonucleotide assembly of part of the variableregion and reconstruction with restriction fragments from other genesoriginally created by SDM and gene assembly14. Expression of CDR-Grafted Genes14.1. Production of Antibody Consisting of Grafted Light (gL) Chainswith Mouse Heavy (mH) or Chimeric Heavy (cH) Chains

All gL chains, in association with mH or cH produced reasonable amountsof antibody. Insertion of the Kozak consensus sequence at a position 5′to the ATG (kgL constructs) however, led to a 2-5 fold improvement innet expression. Over an extended series of experiments expression levelswere raised from approximately 200 ng/ml to approximately 500 ng/ml forkgL/cH or kgL/mH combinations.

When direct binding to antigen on HUT 78 cells was measured, a constructdesigned to include mouse sequence based on loop length (gL121) did notlead to active antibody in association with mH or cH. A constructdesigned to include mouse sequence based on Kabat CDRs (gL221)demonstrated some weak binding in association with mH or cH. However,when framework residues 1, 3, 46, 47 were changed from the human to themurine OKT3 equivalents based on the arguments outlined in Section 12.1antigen binding was demonstrated when both of the new constructs, whichwere termed 121A and 221A were co-expressed with cH. When the effects ofthese residues were examined in more detail, it appears that residues 1and 3 are not major contributing residues as the product of the gL221Bgene shows little detectable binding activity in association with cH.The light chain product of gL221C, in which mouse sequences are presentat 46 and 47, shows good binding activity in association with cH.

14.2 Production of Antibody Consisting of Grafted Heavy (gH) Chains withMouse Light (mL) or Chimeric Light (cL) Chains

Expression of the gH genes proved to be more difficult to achieve thanfor gL. First, inclusion of the Kozak sequence appeared to have nomarked effect on expression of gH genes. Expression appears to beslightly improved but not to the same degree as seen for the graftedlight chain.

Also, it proved difficult to demonstrate production of expectedquantities of material when the loop choice (amino acid 26-32) for CDR1is used, e.g. gH121, 131, 141 an no conclusions can be drawn about theseconstructs.

Moreover, co-expression of the gH341 gene with cL or mL has beenvariable and has tended to produce lower amounts of antibody than thecH/cL or mH/mL combinations. The alterations to gH341 to produce gH341Aand gH341B lead to improved levels of expression.

This may be due either to a general increase in the fraction of mousesequence in the variable region, or to the alteration at position 63where the residue is returned to the human amino acid Valine (Val) fromPhenylalanine (Phe) to avoid possible internal packing problems with therest of the human framework. This arrangement also occurs in gH331 andgH321.

When gH321 or gH331 were expressed in association with cL, antibody wasproduced but antibody binding activity was not detected.

When the more conservative gH341 gene was used antigen binding could bedetected in association with cL or mL, but the activity was onlymarginally above the background level.

When further mouse residues were substituted based on the arguments in12.1, antigen binding could be clearly demonstrated for the antibodyproduced when kgH341A and kgH341B were expressed in association with cL.

14.3 Production of Fully CDR-Grafted Antibody

The kgL221A gene was co-expressed with kgH341, kgH341A or kgH341B. Forthe combination kgH221A/kgH341 very little material was produced in anormal COS cell expression. For the combinations kgL221A/kgH341A orkgH221A/kgH341B amounts of antibody similar to gL/cH was produced.

In several experiments no antigen binding activity could be detectedwith kgH221A/gH341 or kgH221A/kgH341 combinations, although expressionlevels were very low.

Antigen binding was detected when kgL221A/kgH341A or kgH221A/kgH341Bcombinations were expressed. In the case of the antibody produced fromthe kgL221A/kgH341A combination the antigen binding was very similar tothat of the chimeric antibody.

An analysis of the above results is given below.

15. Discussion of CDR-Grafting Results

In the design of the fully humanised antibody the aim was to transferthe minimum number of mouse amino acids that would confer antigenbinding onto a human antibody framework.

15.1. Light Chain

15.1.1. Extent of the CDRs

For the light chain the regions defining the loops known from structuralstudies of other antibodies to contain the antigen contacting residues,and those hypervariable sequences defined by Kabat et al (refs. 4 and 5)as Complementarity Determining Regions (CDRs) are equivalent for CDR2.For CDR1 the hypervariable region extends from residues 24-34 inclusivewhile the structural loop extends from 26-32 inclusive. In the case ofOKT3 there is only one amino acid difference between the two options, atamino acid 24, where the mouse sequence is a serine and the humanframework RE1 has glutamine. For CDR3 the loop extends from residues91-96 inclusive while the Kabat hypervariability extends from residues89-97 inclusive. For OKT3 amino acids 89, 90 and 97 are the same betweenOKT3 and RE1 (FIG. 3). When constructs based on the loop choice for CDR1(gL121) and the Kabat choice (gL221) were made and co-expressed with mHor cH no evidence for antigen binding activity could be found for gL121,but trace activity could be detected for the gL221, suggesting that asingle extra mouse residue in the grafted variable region could havesome detectable effect. Both gene constructs were reasonably wellexpressed in the transient expression system.

15.1.2. Framework Residues

The remaining framework residues were then further examined, inparticular amino acids known from X-ray analysis of other antibodies tobe close to the CDRs and also those amino acids which in OKT3 showeddifferences from the consensus framework for the mouse subgroup(subgroup VI) to which OKT3 shows most homology. Four positions 1, 3, 46and 47 were identified and their possible contribution was examined bysubstituting the mouse amino acid for the human amino acid at eachposition. Therefore gL221A (gL221+D1Q, Q3V, L46R, L47W, see FIG. 3 andTable 1) was made, cloned in EE6hCMVneo and co-expressed with cH(pJA144). The resultant antibody was well expressed and showed goodbinding activity. When the related genes gL221B (gL221+D1Q, Q3V) andgL221C (gL221+L46R, L47W) were made and similarly tested, while bothgenes produced antibody when co-expressed with cH, only the gL221C/cHcombination showed good antigen binding. When the gL121A (gL121+D1Q,Q3V, L46R, L47W) gene was made and co-expressed with cH, antibody wasproduced which also bound to antigen.

15.2. Heavy Chain

15.2.1. Extent of the CDRs

For the heavy chain the loop and hypervariability analyses agree only inCDR3. For CDR1 the loop region extends from residues 26-32 inclusivewhereas the Kabat CDR extends from residues 31-35 inclusive. For CDR2the loop region is from 50-58 inclusive while the hypervariable regioncovers amino acids 50-65 inclusive. Therefore humanised heavy chainswere constructed using the framework from antibody KOL and with variouscombinations of these CDR choices, including a shorter choice for CDR2of 50-56 inclusive as there was some uncertainty as to the definition ofthe end point for the CDR2 loop around residues 56 to 58. The genes wereco-expressed with mL or cL initially. In the case of the gH genes withloop choices for CDR1 e.g. gH121, gH131, gH141 very little antibody wasproduced in the culture supernatants. As no free light chain wasdetected it was presumed that the antibody was being made and assembledinside the cell but that the heavy chain was aberrant in some way,possibly incorrectly folded, and therefore the antibody was beingdegraded internally. In some experiments trace amounts of antibody couldbe detected in ³⁵S labelling studies.

As no net antibody was produced, analysis of these constructs was notpursued further.

When, however, a combination of the loop choice and the Kabat choice forCDR1 was tested (mouse amino acids 26-35 inclusive) and in whichresidues 31 (Ser to Arg), 33 (Ala to Thr), and 35 (Tyr to His) werechanged from the human residues to the mouse residue and compared to thefirst series, antibody was produced for gH321, kgH331 and kgH341 whenco-expressed with cL. Expression was generally low and could not bemarkedly improved by the insertion of the Kozak consensus sequence 5′ tothe ATG of the signal sequence of the gene, as distinct from the case ofthe gL genes where such insertion led to a 2-5 fold increase in netantibody production. However, only in the case of gH341/mL or kgH341/cLcould marginal antigen binding activity be demonstrated. When the kgH341gene was co-expressed with kgL221A, the net yield of antibody was toolow to give a signal above the background level in the antigen bindingassay.

15.2.2. Framework Residues

As in the case of the light chain the heavy chain frameworks werere-examined. Possibly because of the lower initial homology between themouse and human heavy variable domains compared to the light chains,more amino acid positions proved to be of interest. Two genes kgH341Aand kgH341B were constructed, with 11 or 8 human residues respectivelysubstituted by mouse residues compared to gH341, and with the CDR2residue 63 returned to the human amino acid potentially to improvedomain packing. Both showed antigen binding when combined with cL orkgL221A, the kgH341A gene with all 11 changes appearing to be thesuperior choice.

15.3 Interim Conclusions

It has been demonstrated, therefore, for OKT3 that to transfer antigenbinding ability to the humanised antibody, mouse residues outside theCDR regions defined by the Kabat hypervariability or structural loopchoices are required for both the light and heavy chains. Fewer extraresidues are needed for the light chain, possibly due to the higherinitial homology between the mouse and human kappa variable regions.

Of the changes seven (1 and 3 from the light chain and 6, 23, 71, 73 and76 from the heavy chain) are predicted from a knowledge of otherantibody structures to be either partly exposed or on the antibodysurface. It has been shown here that residues 1 and 3 in the light chainare not absolutely required to be the mouse sequence; and for the heavychain the gH341B heavy chain in combination with the 221A light chaingenerated only weak binding activity. Therefore the presence of the 6,23 and 24 changes are important to maintain a binding affinity similarto that of the murine antibody. It was important, therefore, to furtherstudy the individual contribution of othe other 8 mouse residues of thekgH341A gene compared to kgH341.

16. Further CDR-Grafting Experiments

Additional CDR-grafted heavy chain genes were prepared substantially asdescribed above. With reference to Table 2 the further heavy chain geneswere based upon the gh341 (plasmid pJA178) and gH341A (plasmid pJA185)with either mouse OKT3 or human KOL residues at 6, 23, 24, 48, 49, 63,71, 73, 76, 78, 88 and 91, as indicated. The CDR-grafted light chaingenes used in these further experiments were gL221, gL221A, gL221B andgL221C as described above.

TABLE 2 OKT3 HEAVY CHAIN CDR GRAFTS 1. gH341 and derivatives RES NUM6 23 24 48 49 63 71 73 76 78 88 91 OKT3vhQ  K  A  I  G  F  T  K  S  A  A  Y gH341E  S  S  V  A  F  R  N  N  L  G  F JA178 gH341AQ  K  A  I  G  V  T  K  S  A  A  Y JA185 gH341EQ  K  A  I  G  V  T  K  S  A  G  G JA198 gH341*Q  K  A  I  G  V  T  K  N  A  G  F JA207 gH341*Q  K  A  I  G  V  R  N  N  A  G  F JA209 gH341DQ  K  A  I  G  V  T  K  N  L  G  F JA197 gH341*Q  K  A  I  G  V  R  N  N  L  G  F JA199 gH341CQ  K  A  V  A  F  R  N  N  L  G  F JA184 gH341*Q  S  A  I  G  V  T  K  S  A  A  Y JA203 gH341*E  S  A  I  G  V  T  K  S  A  A  Y JA205 gH341BE  S  S  I  G  V  T  K  S  A  A  Y JA183 gH341*Q  S  A  I  G  V  T  K  S  A  G  F JA204 gH341*E  S  A  I  G  V  T  K  S  A  G  F JA206 gH341*Q  S  A  I  G  V  T  K  N  A  G  F JA208 KOLE  S  S  V  A     R  N  N  L  G  F OKT3 LIGHT CHAIN CDR GRAFTS2. gL221 and derivatives (SEQ ID NO: 7, 10, and 11-24) RES NUM 1 3 46 47OKT3v1 Q V R   W GL221 D Q L   L DA221 gL221A Q V R   W DA221A gL221BQ V L   L DA221B GL221C D Q R   W DA221C RE1 D Q L   L(SEQ ID NO: 5, 8, 9, and 25-28)

The CDR-grafted heavy and light chain genes were co-expressed in COScells either with one another in various combinations but also with thecorresponding murine and chimeric heavy and light chain genessubstantially as described above. The resultant antibody products werethen assayed in binding and blocking assays with HPB-ALL cells asdescribed above.

The results of the assays for various grafted heavy chains co-expressedwith the gL221C light chain are given in FIGS. 7 and 8 (for the JA184,JA185, JA197 and JA198 constructs—see Table 2), in FIG. 9 (for theJA183, JA184, JA185 and JA197 constructs) in FIGS. 10a and b (for thechimeric, JA185, JA199, JA204, JA205, JA207, JA208 and JA209 constructs)and in FIGS. 11a and b (for the JA183, JA184, JA185, JA198, JA203, JA205and JA206 constructs).

The basic grafted product without any human to murine changes in thevariable frameworks, i.e. gL221 co-expressed with gh341 (JA178), andalso the “fully grafted” product, having most human to murine changes inthe grafted heavy chain framework, i.e. gL221C co-expressed with gh341A(JA185), were assayed for relative binding affinity in a competitionassay against murine OKT3 reference standard, using HPB-ALL cells. Theassay used was as described above in section 3.3. The results obtainedare given in FIG. 12 for the basic grafted product and in FIG. 13 forthe fully grafted product. These results indicate that the basic graftedproduct has neglibible binding ability as compared with the OKT3 murinereference standard; whereas the “fully grafted” product has a bindingability very similar to that of the OKT3 murine reference standard.

The binding and blocking assay results indicate the following:

The JA198 and JA207 constructs appear to have the best bindingcharacteristics and similar binding abilities, both substantially thesame as the chimeric and fully grafted gH341A products. This indicatesthat positions 88 and 91 and position 76 are not highly critical formaintaining the OKT3 binding ability; whereas at least some of positions6, 23, 24, 48, 49, 71, 73 and 78 are more important.

This is borne out by the finding that the JA209 and JA199, although ofsimilar binding ability to one another, are of lower binding abilitythan the JA198 and JA207 constructs. This indicates the importance ofhaving mouse residues at positions 71, 73 and 78, which are eithercompletely or partially human in the JA199 and JA209 constructsrespectively.

Moreover, on comparing the results obtained for the JA205 and JA183constructs it is seen that there is a decrease in binding going from theJA205 to the JA183 constructs. This indicates the importance ofretaining a mouse residue at positions 23, the only position changedbetween JA205 and JA183.

These and other results lead us to the conclusion that of the 11 mouseframework residues used in the gH341A (JA185) construct, it is importantto retain mouse residues at all of positions 6, 23, 24, 48 and 49, andpossibly for maximum binding affinity at 71, 73 and 78.

Similar Experiments were carried out to CDR-graft a number of the rodentantibodies including antibodies having specificity for CD4 (OKT4),ICAM-1 (R6-5), TAG72 (B72.3), and TNF α(61E71, 101.4, hTNF1, hTNF2 andhTNF3).

Example 2

CDR-Grafting of a Murine Anti-CD4 T Cell Receptor Antibody, OKT4A

Anti OKT4A CDR-grafted heavy and light chain genes were prepared,expressed and tested substantially as described above in Example 1 forCDR-grafted OKT3. The CDR grafting of OKT4A is described in detail inOrtho patent application PCT/GB 90 . . . of even date herewith entitled“Humanised Antibodies”. The disclosure of this Ortho patent applicationPCT/GB 90 . . . is incorporated herein by reference. A number ofCDR-grafted OKT4 antibodies have been prepared. Presently theCDR-grafted OKT4A of choice is the combination of the grafted lightchain LCDR2 and the grafted heavy chain HCDR10.

The Light Chain

The human acceptor framework used for the grafted light chains was RE1.The preferred LCDR2 light chain has human to mouse changes at positions33, 34, 38, 49 and 89 in addition to the structural loop CDRs. Of thesechanged positions, positions 33, 34 and 89 fall within the preferredextended CDRs of the present invention (positions 33 and 34 in CDR1 andposition 89 in CDR3). The human to murine changes at positions 38 and 49corresponds to positions at which the amino acid residues are preferablydonor murine amino acid residues in accordance with the presentinvention. A comparison of the amino acid sequences of the donor murinelight chain variable domain and the RE1 human acceptor light chainvariable further reveals that the murine and human residues areidentical at all of positions 46, 48 and 71 and at all of positions 2,4, 6, 35, 36, 44, 47, 62, 64-69, 85, 87, 98, 99 and 101 and 102. Howeverthe amino acid residue at position 58 in LCDR2 is the human RE1framework residue not the mouse OKT4 residue as would be preferred inaccordance with the present invention.

The Heavy Chain

The human acceptor framework used for the grafted heavy chains was KOL.

The preferred CDR graft HCDR10 heavy chain has human to mouse changes atpositions 24, 35, 57, 58, 60, 88 and 91 in addition to the structuralloop CDRs.

Of these positions, positions 35 (CDR1) and positions 57, 58 and 60(CDR2) fall within the preferred extended CDRs of the present invention.Also the human to mouse change at position 24 corresponds to a positionat which the amino acid residue is a donor murine residue in accordancewith the present invention. Moreover, the human to mouse changes atpositions 88 and 91 correspond to positions at which the amino acidresidues are optionally donor murine residues.

Moreover, a comparison of the murine OKT4A and human KOL heavy chainvariable amino acid sequences reveals that the murine and human residuesare identical at all of positions 23, 49, 71, 73 and 78 and at all ofpositions 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and107.

Thus the OKT4A CDR-grafted heavy chain HCDR10 corresponds to aparticularly preferred embodiment according to the present invention.

Example 3

CDR-Grafting of an Anti-Mucin Specific Murine Antibody, B72.3

The cloning of the genes coding for the anti-mucin specific murinemonoclonal antibody B72.3 and the preparation of B72.3 mouse-humanchimeric antibodies has been described previously (ref 13 and WO89/01783). CDR-grafted versions of B72.3 were prepared as follows.

-   (a) B72.3 Light Chain    -   CDR-grafting of this light chain was accomplished by direct        transfer of the murine CDRs into the framework of the human        light chain RE1. The regions transferred were:

CDR Number Residues 1 24-34 2 50-56 3 90-96

-   -    The activity of the resulting grafted light chain was assessed        by co-expression in COS cells, of genes for the combinations:        -   B72.3 cH/B72.3 cL    -   and        -   B72.3 cH/B72.3 gL    -    Supernatants were assayed for antibody concentration and for        the ability to bind to microtiter plates coated with mucin. The        results obtained indicated that, in combination with the B72.3        cH chain, B72.3 cL and B72.3 gL had similar binding properties.

Comparison of the murine B72.3 and REI light chain amino acid sequencereveals that the residues are identical at positions 46, 58 and 71 butare different at positions 48.

Thus changing the human residue to the donor mouse residue at position48 may further improve the binding characteristics of the CDR-graftedlight chain, (B72.3 gL) in accordance with the present invention.

-   (b) B72.3 heavy chain    -   i. Choice of framework    -   At the outset it was necessary to make a choice of human        framework. Simply put, the question was as follows: Was it        necessary to use the framework regions from an antibody whose        crystal structure was known or could the choice be made on some        other criteria?    -   For B72.3 heavy chain, it was reasoned that, while knowledge of        structure was important, transfer of the CDRs from mouse to        human frameworks might be facilitated if the overall homology        between the donor and receptor frameworks was maximised.        Comparison of the B72.3 heavy chain sequence with those in Kabat        (ref. 4) for human heavy chains showed clearly that B72.3 had        poor homology for KOL and NEWM (for which crystal structures are        available) but was very homologous to the heavy chain for EU.    -   On this basis, EU was chosen for the CDR-grafting and the        following residues transferred as CDRs.

CDR Number Residues 1 27-36 2 50-63 3  93-102

Also it was noticed that the FR4 region of EU was unlike that of anyother human (or mouse) antibody. Consequently, in the grafted heavychain genes this was also changed to produce a “consensus” humansequence. (Preliminary experiments showed that grafted heavy chain genescontaining the EU FR4 sequence expressed very poorly in transientexpression systems.)

-   -   ii. Results with grafted heavy chain genes    -   Expression of grafted heavy chain genes containing all human        framework regions with either gL or cL genes produced a grafted        antibody with little ability to bind to mucin. The grafted        antibody had about 1% the activity of the chimeric antibody. In        these experiments, however, it was noted that the activity of        the grafted antibody could be increased to ˜10% of B72.3 by        exposure to pHs of 2-3.5.    -   This observation provided a clue as to how the activity of the        grafted antibody could be improved without acid treatment. It        was postulated that acid exposure brought about the protonation        of an acidic residue (pKa of aspartic acid=3.86 and of glutamine        acid=4.25) which in turn caused a change in structure of the CDR        loops, or allowed better access of antigen. From comparison of        the sequences of B72.3 (ref. 13) and EU (refs. 4 and 5), it was        clear that, in going from the mouse to human frameworks, only        two positions had been changed in such a way that acidic        residues had been introduced. These positions are at residues 73        and 81, where K to E and Q to E changes had been made,        respectively.    -   Which of these positions might be important was determined by        examining the crystal structure of the KOL antibody. In KOL        heavy chain, position 81 is far removed from either of the CDR        loops.    -   Position 73, however, is close to both CDRs 1 and 3 of the heavy        chain and, in this position it was possible to envisage that a K        to E change in this region could have a detrimental effect on        antigen binding.    -   iii. Framework changes in B72.3 gH gene    -   On the basis of the above analysis, E73 was mutated to a lysine        (K). It was found that this change had a dramatic effect on the        ability of the grafted Ab to bind to mucin. Further the ability        of the grafted B72.3 produced by the mutated gH/gL combination        to bind to mucin was similar to that of the B72.3 chimeric        antibody.    -   iv. Other framework changes    -   In the course of the above experiments, other changes were made        in the heavy chain framework regions. Within the accuracy of the        assays used, none of the changes, either along or together,        appeared beneficial.    -   v. Other    -   All assays used measured the ability of the grafted Ab to bind        to mucin and, as a whole, indicated that the single framework        change at position 73 is sufficient to generate an antibody with        similar binding properties to B72.3.    -   Comparison of the B72.3 murine and EU heavy chain sequences        reveals that the mouse and human residues are identical at        positions 23, 24, 71 and 78.    -   Thus the mutated CDR-grafted B72.3 heavy chain corresponds to a        preferred embodiment of the present invention.

Example 4

CDR-Grafting of a Murine Anti-ICAM-1 Monoclonal Antibody

A murine antibody, R6-5-D6 (EP 0314863) having specificity forIntercellular Adhesion Molecule 1 (ICAM-1) was CDR-grafted substantiallyas described above in previous examples. This work is described ingreater detail in co-pending application, British Patent Application No.9009549.8, the disclosure of which is incorporated herein by reference.

The human EU framework was used as the acceptor framework for both heavyand light chains. The CDR-grafted antibody currently of choice isprovided by co-expression of grafted light chain gL221A and graftedheavy chain gH341D which has a binding affinity for ICAM 1 of about 75%of that of the corresponding mouse-human chimeric antibody.

Light Chain

gL221A has murine CDRs at positions 24-34 (CDR1), 50-56 (CDR2) and 89-97(CDR3). In addition several framework residues are also the murine aminoacid. These residues were chosen after consideration of the possiblecontribution of these residues to domain packing and stability of theconformation of the antigen binding region. The residues which have beenretained as mouse are at positions 2, 3, 48 (7), 60, 84, 85 and 87.Comparison of the murine anti-ICAM 1 and human EU light chain amino acidsequences reveals that the murine and human residues are identical atpositions 46, 58 and 71.

Heavy Chain

gH341D has murine CDRs at positions 26-35 (CDR1), 50-56 (CDR2) and94-100B (CDR3). In addition murine residues were used in gH341D atpositions 24, 48, 69, 71, 73, 80, 88 and 91. Comparison of the murineanti-ICAM 1 and human EU heavy chain amino acid sequences are identicalat positions 23, 49 and 78.

Example 5

CDR-Grafting of Murine Anti-TNFa antibodies

A number of murine anti-TNFa monoclonal antibodies were CDR-graftedsubstantially as described above in previous examples. These antibodiesinclude the murine monoclonal antibodies designated 61 E71, hTNF1, hTNF3and 101.4 A brief summary of the CDR-grafting of each of theseantibodies is given below.

61E71

A similar analysis as described above (Example 1, Section 12.1.) wasdone for 61E71 and for the heavy chain 10 residues were identified at23, 24, 48, 49, 68, 69, 71, 73, 75 and 88 as residues to potentiallyretain as murine. The human frameworks chosen for CDR-grafting of thisantibody, and the hTNF3 and 101.4 antibodies were RE1 for the lightchain and KOL for the heavy chain. Three genes were built, the first ofwhich contained 23, 24, 48, 49, 71 and 73 [gH341(6)] as murine residues.The second gene also had 75 and 88 as murine residues [gH341(8)] whilethe third gene additionally had 68, 69, 75 and 88 as murine residues[gH341(10)]. Each was co-expressed with gL221, the minimum grafted lightchain (CDRs only). The gL221/gH341(6) and gL221/gH341 (8) antibodiesboth bound as well to TNF as murine 61E71. The gL221/gH341(10) antibodydid not express and this combination was not taken further.

Subsequently the gL221/gH341(6) antibody was assessed in an L929 cellcompetition assay in which the antibody competes against the TNFreceptor on L929 cells for binding to TNF in solution. To this assay thegL221/gH341(6) antibody was approximately 10% as active as murine 61E71.

hTNF1

hTNF1 is a monoclonal antibody which recognises an epitope on humanTNF-. The EU human framework was used for CDR-grafting of both the heavyand light variable domains.

Heavy Chain

In the CDR-grafted heavy chain (ghTNF1) mouse CDRs were used atpositions 26-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3). Mouse residueswere also used in the frameworks at positions 48, 67, 69, 71, 73, 76,89, 91, 94 and 108. Comparison of the TNF1 mouse and EU human heavychain residues reveals that these are identical at positions 23, 24, 29and 78.

Light Chain

In the CDR-grafted light chain (gLhTNF1) mouse CDRs wre used atpositions 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3). In addition mouseresidues were used in the frameworks at positions 3, 42, 48, 49, 83, 106and 108. Comparison of the hTNF1 mouse and EU human light chain residuesreveals that these are identical at positions 46, 58 and 71.

The grafted hTNF1 heavy chain was co-expressed with the chimeric lightchain and the binding ability of the product compared with that of thechimeric light chain/chimeric heavy chain product in a TNF bindingassay. The grafted heavy chain product appeared to have binding abilityfor TNF slightly better than the fully chimeric product.

Similarly, a grafted heavy chain/grafted light chain product wasco-expressed and compared with the fully chimeric product and found tohave closely similar binding properties to the latter product.

hTNF3

hTNF3 recognises an epitope on human TNF-α. The sequence of hTNF3 showsonly 21 differences compared to 61E71 in the light and heavy chainvariable regions, 10 in the light chain (2 in the CDRs at positions 50,96 and 8 in the framework at 1, 19, 40, 45, 46, 76, 103 and 106) and 11in the heavy chain (3 in the CDR regions at positions 52, 60 and 95 and8 in the framework at 1, 10, 38, 40, 67, 73, 87 and 105). The light andheavy chains of the 61E71 and hTNF3 chimeric antibodies can be exchangedwithout loss of activity in the direct binding assay. However 61E71 isan order of magnitude less able to compete with the TNF receptor on L929cells for TNF-a compared to hTNF3. Based on the 61E71 CDR grafting datagL221 and gH341(+23, 24, 48, 49 71 and 73 as mouse) genes have beenbuilt for hTNF3 and tested and the resultant grafted antibody binds wellto TNF-a, but competes very poorly in the L929 assay. It is possiblethat in this case also the framework residues identified for OKT3programme may improve the competitive binding ability of this antibody.

101.4

101.4 is a further murine monoclonal antibody able to recognise humanTNF-a. The heavy chain of this antibody shows good homology to KOL andso the CDR-grafting has been based on RE1 for the light chain and KOLfor the heavy chain. Several grafted heavy chain genes have beenconstructed with conservative choices for the CDR's (gH341) and whichhave one or a small number of non-CDR residues at positions 73, 78 or77-79 inclusive, as the mouse amino acids. These have been co-expressedwith cL or gL221. In all cases binding to TNF equivalent to the chimericantibody is seen and when co-expressed with cL the resultant antibodiesare able to compete well in the L929 assay. However, with gL221 theresultant antibodies are at least an order of magnitude less able tocompete for TNF against the TNF receptor on L929 cells.

Mouse residues at other positions in the heavy chain, for example, at 23and 24 together or at 76 have been demonstrated to provide noimprovement to the competitive ability of the grafted antibody in theL929 assay.

A number of other antibodies including antibodies having specificity forinterleukins e.g. IL1 and cancer markers such as carcinoembryonicantigen (CEA) e.g. the monoclonal antibody A5B7 (ref 21), have beensuccessfully CDR-grafted according to the present invention. It will beappreciated that the foregoing examples are given by way of illustrationonly and are not intended to limit the scope of the claimed invention.Changes and modifications may be made to the methods described whilststill falling within the spirit and scope of the invention.

REFERENCES

-   1. Kohler & Milstein, Nature, 265, 295-497, 1975.-   2. Chatenoud et al, (1986), J. Immunol. 137, 830-838.-   3. Jeffers et al, (1986), Transplantation, 41, 572-578.-   4. Begent et al, Br. J. Cancer 62: 487 (1990).-   5. Verhoeyen et al, Science, 239, 1534-1536, 1988.-   6. Riechmann et al, Nature, 332, 323-324, 1988.-   7. Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M.,    Gottesman, K. S., 1987, in Sequences of Proteins of Immunological    Interest, US Department of Health and Human Services, NIH, USA.-   8. Wu, T. T., and Kabat, E. A., 1970, J. Exp. Med. 132 211-250.-   9. Queen et al, (1989), Proc. Natl. Acad. Sci. USA, 86, 10029-10033    and WO 90/07861-   10. Maniatis et al, Molecular Cloning, Cold Spring Harbor, N.Y,    1989.-   11. Primrose and Old, Principles of Gene Manipulation, Blackwell,    Oxford, 1980.-   12. Sanger, F., Nicklen, S., Coulson, A. R., 1977, Proc. Natl. Acad.    Sci. USA, 74 5463-   13. Kramer, W., Drutsa, V., Jansen, H. -W., Kramer, B., Plugfelder,    M., Fritz, H. -J., 1984, Nucl. Acids Res. 12, 9441-   14. Whittle, N., Adair, J., Lloyd, J. C., Jenkins, E., Devine, J.,    Schlom, J., Raubitshek, A., Colcher, D., Bodmer, M., 1987, Protein    Engineering 1, 499.-   15. Sikder, S. S., Akolkar, P. N., Kaledas, P. M., Morrison, S. L.,    Kabat, E. A., 1985, J. Immunol. 135, 4215.-   16. Wallick, S. C., Kabat, E. A., Morrison, S. L., 1988, J. Exp.    Med. 168, 1099-   17. Bebbington, C. R., Published International Patent Application WO    89/01036.-   18. Granthan and Perrin 1986, Immunology Today 7, 160.-   19. Kozak, M., 1987, J. Mol. Biol. 196, 947.-   20. Jones, T. P., Dear, P. H., Foote, J., Neuberger, M. S., Winter,    G., 1986, Nature, 321, 522-   21. Harwood et al, Br. J. Cancer, 54, 75-82 (1986).

The invention claimed is:
 1. A humanised antibody molecule having affinity for an antigen and comprising a composite heavy chain and a complementary light chain, said composite heavy chain having a variable domain including complementarity determining regions (CDRs), wherein, according to the Kabat numbering system, in said composite heavy chain at least residues 26 to 35, 50 to 58 and 95 to 102 in the CDRs and at least residues 48, 49, 71, 73, 76, 78, 88, and 91 in the framework regions are non-human donor, provided that said heavy chain is not a chimeric antibody heavy chain having a donor variable domain and a human constant domain.
 2. A humanised antibody molecule having affinity for a predetermined antigen and comprising a composite heavy chain and a complementary light chain, said composite heavy chain having a variable domain including complementarity determining regions (CDRs) and framework regions, wherein, according to the Kabat numbering system, in said composite heavy chain: said CDRs are non-human donor at residues 31 to 35, 50 to 58, and 95 to 102; and said framework regions are non-human donor at: a) residue 6; b) one or more of residues 23 and 24; c) one or more of residues 48 and 49; d) one or more of residues 71 and 73; e) residue 75; f) one or more of residues 75, 76, and 78 76 and 78; and f)g) one or more of residues 88 and 91, provided that said heavy chain is not a chimeric antibody heavy chain having a donor variable domain and a human constant domain.
 3. The antibody molecule of claim 2 wherein residue 2 of said composite heavy chain is donor.
 4. The antibody molecule of claim 2 wherein residue 72 of said composite heavy chain is donor.
 5. The antibody molecule of claim 2 wherein residue 108 of said composite heavy chain is donor.
 6. The antibody molecule of claim 2 wherein residue 110 of said composite heavy chain is donor.
 7. A humanised antibody molecule having affinity for an antigen and comprising a composite heavy chain and a complementary light chain, said composite heavy chain having a variable domain including complementarity determining regions (CDRs), wherein, according to the Kabat numbering system, in said composite heavy chain at least residues 31 to 35, 50 to 58 and 95 to 102 in the CDRs, and at least residues 6, 24, 48, 49, 71, 72, 73, and 78 in the framework regions are non-human donor, provided that said heavy chain is not a chimeric antibody heavy chain having a donor variable domain and a human constant domain.
 8. A humanised antibody molecule having affinity for an antigen and comprising a composite heavy chain and a complementary light chain, said composite heavy chain having a variable domain including complementarity determining regions (CDRs), wherein, according to the Kabat numbering system, in said composite heavy chain at least residues 31 to 35, 50 to 58 and 95 to 102 in the CDRs, and at least residues 6, 24, 48, 49, 71, 73, 78, and 108 in the framework regions are non-human donor, provided that said heavy chain is not a chimeric antibody heavy chain having a donor variable domain and a human constant domain.
 9. A humanised antibody molecule having affinity for an antigen and comprising a composite heavy chain and a complementary light chain, said composite heavy chain having a variable domain including complementarity determining regions (CDRs), wherein, according to the Kabat numbering system, in said composite heavy chain at least residues 31 to 35, 50 to 58 and 95 to 102 in the CDRs, and at least residues 6, 24, 48, 49, 71, 73, 78, and 110 in the framework regions are non-human donor, provided that said heavy chain is not a chimeric antibody heavy chain having a donor variable domain and a human constant domain.
 10. A humanised antibody molecule having affinity for an antigen and comprising a composite heavy chain and a complementary light chain, said composite heavy chain having a variable domain including complementarity determining regions (CDRs), wherein, according to the Kabat numbering system, in said composite heavy chain at least residues 31 to 35, 50 to 58 and 95 to 102 in the CDRs, and at least residues 6, 24, 48, 49, 71, 73, 76, 78, 88, and 91 in the framework regions are non-human donor, provided that said heavy chain is not a chimeric antibody heavy chain having a donor variable domain and a human constant domain.
 11. The humanised antibody molecule of claim 10, wherein residue 2 of said composite heavy chain is donor.
 12. The humanised antibody molecule of claim 10, wherein residue 72 of said composite heavy chain is donor.
 13. The humanised antibody molecule of claim 10, wherein residue 108 of said composite heavy chain is donor.
 14. The humanised antibody molecule of claim 10, wherein residue 110 of said composite heavy chain is donor.
 15. A humanised antibody molecule having affinity for an antigen and comprising a composite heavy chain and a complementary light chain, said composite heavy chain having a variable domain including complementarity determining regions (CDRs), wherein, according to the Kabat numbering system, in said composite heavy chain at least residues 31 to 35, 50 to 58 and 95 to 102 in the CDRs, and at least residues 6, 24, 48, 49, 71, 73, 76, and 78 in the framework regions are non-human donor, provided that said heavy chain is not a chimeric antibody heavy chain having a donor variable domain and a human constant domain.
 16. The humanised antibody molecule of claim 15, wherein residue 2 of said composite heavy chain is donor.
 17. The humanised antibody molecule of claim 15, wherein residue 72 of said composite heavy chain is donor.
 18. The humanised antibody molecule of claim 15, wherein residue 108 of said composite heavy chain is donor.
 19. The humanised antibody molecule of claim 15, wherein residue 110 of said composite heavy chain is donor. 